HPLC/UPLC Cleaning Protocol Materials HPLC grade Isopropanol HPLC grade acetonitrile HPLC grade triflouroacetic acid
Milipore H2O Trifluouroethanol Solutions A. 0.1 % Triflouroacetic acid in Water Prepare water by filtering Millipore water through a 0.2 um nylon filter. Degas for 20 min by putting it in sonicator bath under a vacuum. Add 0.1% V/V triflouroacetic acid with 1ml glass pippette. B. 0.1% Triflouroacetic acid in 75% isopropanol 25% acetonitrile. Add 250ml of acetonitrile and 750 ml of isopropanol into a 1L glass bottle. Add 1 ml of Triflouroacetic acid. ** If triflouroacetic acid is brown instead of clear discard the bottle. C. Trifluoroethanol Filter triflouoethanol through 0.2 um Titan PTFE syringe filter into a 1.5ml autosampler vial and cap Running Method: 0.4ml/min 80 deg C 100 ul or 20 ul (maximum) injection of trifluoroethanol 0-5min 75%A 25%B 5-35min gradient to 100%B 35-40 100%B stop time at 40 min 5 min post time return to initial conditions Running procedure Turn column in the reverse direction and set it up to flow into a beaker. Screw an A inlet line into solution A and B inlet line into solution B. Be sure the running method reflects the proper solution positions. Purge the instrument with the cleaning solutions for appropriate length of time to flush the system ~10 min HPLC and ~3min UPLC.
1 ml/min and bring to .Gradually start the flow in the reverse direction. Start the flow at .
.1 ml/min gradually increasing to normal running flow rate. Program 3 runs of the method with an injection of trifluouroethanol each time. Start at . Allow column to equilibrate for 30 min before running a check standard.4 over several minutes. Allow the column to cool before unhooking. At the end of the three runs set the flow to 0 ml/min and the thermostat to 30 deg or less. Screw the usual running solvents back into the inlet lines. Purge the system again. Start the running method when the temperature reaches 70-80 deg. Return the column to the normal operating direction. Try to rinse off the lines a bit and wipe with a Kim wipe to minimize carryover. Run for 10 minutes before stopping the flow and hooking up to the detector again.